Genome engineering using the bacterial RNA-guided CRISPR-Cas9 immune system in animals and plants is transforming biology. Efforts from its discovery through the elucidation of the enzyme mechanism are providing the foundation for remarkable developments to modify, regulate or mark genomic loci in a wide variety of cells and organisms. CRISPR-Cas9 gene editing has the potential to transform medicine by providing innovative ways to probe biology and treat genetic disorders in adults. The key goals of the proposed project are: 1) to train in the new fields of biochemistry and structural biology (K99 phase), 2) to characterize and develop novel Cas9 variants for biomedical applications (K99/R00 phase), and 3) to establish a successful, independent research laboratory at a leading academic institution (R00 phase). My laboratory will be focused on mechanism and therapeutic application of RNA-guided immune systems, to address the urgent medical need for new platform technologies to establish diverse therapeutic targets and develop innovative treatment modalities, and to mentor the next generation of scientists. Candidate: I am committed to an academic career in biomedical research. My long-term goals are to secure a tenure-track faculty position at a leading academic institution and successfully establish an independent research laboratory. My work focuses on mechanism and therapeutic application of RNA-guided immune systems. My multidisciplinary training in molecular cell biology and in-vivo models of human disease, combined with the new biochemical and structural approaches learned during the K99 mentored phase, will allow me to bridge basic science and patient-focused research. In turn, this will allow me to help accelerate translational approaches and develop novel treatment paradigms for genetic disorders. Importantly, I am committed to mentor the next generation of students and serve as a role model to help transform talented young scientist into successful faculty members. Environment: The mentored phase (K99) will be carried out in the laboratory of Dr. Jennifer A. Doudna at UC Berkeley, a highly interactive and vibrant research environment, to learn new skills in biochemistry and structural biology. The Doudna Laboratory is located at Stanley Hall, which serves as the UC Berkeley hub for the California Institute for Quantitative Bioscience (QB3). The Doudna lab has many multidisciplinary interactions with the more than 200 researchers that are part of QB3, involving the the biological sciences, chemical sciences, physical sciences and engineering. These collaborations result in enhanced access to cutting-edge expertise in biochemistry, structural biology, biophysics, computational modeling, high-throughput sequencing and large-scale data analysis. I will benefit from this vast resource of talent and knowledge, as well as access to the specialized equipment needed to carry out the proposed research. Importantly, Dr. Doudna is a leading expert in biochemistry and structural biology, and a pioneer of the CRISPR-Cas9 genome editing technology. Research: CRISPR-Cas9 gene editing has the potential to enhance medicine by providing innovative ways to probe biology and treat genetic disorders in adult patients. The goal of this project is to define mechanisms and establish novel Cas9 variants for efficient CRISPR-Cas9 mediated genome editing in vivo. To this end, we will combine computational, biochemical and high-throughput cell-based approaches. The Specific Aims of this proposal are: Aim 1) to characterize novel Cas9 endonucleases, and Aim 2) to establish a DNA-guided CRISPR system for efficient in-vivo genome editing. Completion of the proposed project will result in new platform technologies and applications for efficient CRISPR-based gene editing, to meet the urgent medical need for new tools to accelerate drug discovery and develop innovative treatment modalities. 1) Newly defined Cas9 enzymes will yield orthogonal CRISPR systems that can be used in parallel for multiplexed genome editing. These tools will allow probing biology with unprecedented precision and speed, and expand our understanding of homeostasis and disease. In turn, this will lead to better treatments for patients. Additionally, ssDNA or ss/dsRNA targeting Cas9s may lead to new methods to assess genomes/transcriptomes and can provide novel insight into the mechanisms of bacterial immunity. 2) The DNA-guided Cas9 version will facilitate therapeutic applications by overcoming the bottleneck of sgRNA instability in serum, and constitute the basis for the development of innovative CRISPR-based strategies to treat genetic disorders. Importantly, the methods learned and data generated during the mentored phase of the award (K99) will provide me with the foundation for future projects and grants (R00 and beyond). Together with my expertise in cell biology and animal modeling, this will allow me to successfully establish an independent laboratory at a leading academic research institution.